Non-insulin-dependent diabetes mellitus (NIDDM) is characterized by insulin resistance of the peripheral tissues, including the skeletal muscle, liver, and adipose. The resulting hyperglycemia is often accompanied by defective lipid metabolism which can lead to cardiovascular complications such as atherosclerosis and hypertension.
Thiazolidinediones comprise a group of structurally-related antidiabetic compounds that increases the insulin sensitivity of target tissues (skeletal muscle, liver, adipose) in insulin resistant animals. In addition to these effects on hyperglycemia, thiazolidinediones also reduce lipid and insulin levels in animal models of NIDDM. Recently, the thiazolidinedione troglitazone was shown to have these same beneficial effects in human patients suffering from impaired glucose tolerance, a metabolic condition that precedes the development of NIDDM, as in patients suffering from NIDDM (Nolan et al., 1994). While their mechanism of action remains unclear, it is known that the thiazolidinediones do not cause increases in insulin secretion or in the number or affinity of insulin receptor binding sites, suggesting that thiazolidinediones amplify post-receptor events in the insulin signaling cascade (Colca and Morton, 1990, Chang et al., 1983).
Thiazolidinediones have been found to be efficacious inducers of differentiation in cultured pre-adipocyte cell lines (Hiragun et al., 1988; Sparks et al., 1991; Kletzien et al., 1992). Treatment of pre-adipocyte cell lines with the thiazolidinedione pioglitazone results in increased expression of the adipocyte-specific genes aP2 and adipsin as well as the glucose transporter proteins GLUT-1 and GLUT-4. These data suggest that the hypoglycemic effects of thiazolidinediones seen in vivo may be mediated through adipose tissue. However, as estimates of the contribution of adipose tissue to whole body glucose usage range from only 1-3%, it remains unclear whether the hypoglycemic effects of thiazolidinediones can be accounted for by changes in adipocytes. Additionally, thiazolidinediones have been implicated in appetite regulation disorders, see PCT Patent application WO 94/25026 A1, and in increase of bone marrow fat content, (Williams, et al, 1993).
Peroxisome proliferator-activated receptor gamma (PPAR.gamma.) is an orphan member of the steroid/thyroid/retinoid superfamily of ligand-activated transcription factors. PPAR.gamma. is one of a subfamily of closely-related PPARs encoded by independent genes (Dreyer et al., 1992; Schmidt et al, 1992; Zhu et al., 1993; Kliewer et al., 1994). Three mammalian PPARs have been identified and termed PPAR.alpha.,.gamma., and NUC-1. Homologs of PPAR.alpha. and .gamma. have been identified in the frog, Xenopus laevis; however, a third Xenopus PPAR, termed PPAR.beta., is not a NUC-1 homolog, leading to the suggestion that there may be additional subtypes in either or both species.
The PPARs are activated to various degrees by high (micromolar) concentrations of long-chain fatty acids and peroxisome proliferators (Isseman and Green, 1990; Gottlicher, 1992). Peroxisome proliferators are a structurally diverse group of compounds that includes herbicides, phthalate plasticizers, and the fibrate class of hypolipidemic drugs. While these data suggest that the PPARs are bona fide receptors, they remain "orphans" as none of these compounds have been shown to interact directly with the PPARs.
PPARs regulate expression of target genes by binding to DNA sequence elements, termed PPAR response elements (PPRE), as heterodimers with the retinoid X receptors (reviewed in Keller and Whali, 1993). To date, PPREs have been identified in the enhancers of a number of genes encoding proteins that regulate lipid metabolism including the three enzymes required for peroxisomal beta-oxidation of fatty acids, medium-chain acyl-CoA dehydrogenase, a key enzyme in mitochondrial beta-oxidation, and aP2, a lipid binding protein expressed exclusively in adipocytes. The nature of the PPAR target genes coupled with the activation of PPARs by fatty acids and hypolipidemic drugs suggests a physiological role for the PPARs in lipid homeostasis (reviewed in Keller and Whali, 1993).
Recently, a second isoform of PPAR.gamma., termed PPAR.gamma.2, was cloned from a mouse adipocyte library (Tontonoz et al., 1994). PPAR.gamma.1 and .gamma.2 differ in only 30 amino acids at the extreme N-terminus of the receptor and likely arise from a single gene. PPAR.gamma.2 is expressed in a strikingly adipose-specific manner and its expression is markedly induced during the course of differentiation of several preadipocyte cell lines; furthermore, forced expression of PPAR.gamma.2 was shown to be sufficient to activate the adipocyte-specific aP2 enhancer in non-adipocyte cell lines. These data suggest that PPAR.gamma.2 plays an important role in adipocyte differentiation.
Recently, the thiazolidinedione pioglitazone was reported to stimulate expression of a chimeric gene containing the enhancer/promoter of the lipid-binding protein aP2 upstream of the chloroamphenicol acetyl transferase reporter gene (Harris and Kletzien, 1994). Deletion analysis led to the identification of an approximately 30 bp region responsible for pioglitazone responsiveness. Interestingly, in an independent study, this 30 bp fragment was shown to contain a PPRE (Tontonoz et al., 1994). Taken together, these studies suggested the possibility that the thiazolidinediones modulate gene expression at the transcriptional level through interactions with a PPAR.